Vaccinia virus presents a model system in which to study virus multiplication, pathogenesis, and regulatory mechanisms. The usefulness of a genetic approach to study viral replication has been established by temperature-sensitive (ts) mutants of bacteriophage and animal viruses. Because of the large size of the vaccinia genome, a successful genetic analysis will require a large number of mutants in different genes as determined by complementation experiments and biochemical characterization of their ts defects. In addition, the mutants must have low levels of reversion and leakiness. The primary aim of this research proposal is to isolate ts mutants of vaccinia virus and to determine their usefulness for genetic and biochemical studies according to the above criteria. The wild-type virus will be mutagenized with 5-bromodeoxyuridine, nitrosoguanidine, and nitrous acid. Initially, plaques will be picked at random from monolayers infected with mutagenized virus at the permissive temperature and replaqued at the high and low temperatures. In later experiments, selection techniques will be employed to increase the frequency of mutant isolation. Once mutants have been isolated, the number of genes represented will be determined by complementation analysis. The usefulness of rapid qualitative complementation techniques will be investigated. Finally, the temperature-sensitive defects of the mutants will be determined.